Transglutaminases are Ca2+ dependent enzymes that catalyze the formation of isopeptide bonds in proteins between the side chain γ-carboxamide group of glutamine and the side chain ε-amino group of lysine. Transglutaminases are found both extracellularly and intracellularly. Examples of transglutaminases include Factor XIII, epidermal transglutaminases, type II transglutaminases (tissue type transglutaminase, liver transglutaminases), and type I transglutaminases (keratinocyte transglutaminase).
Blood coagulation Factor XIII (FXIII) is a plasma transglutaminase also known as “fibrinoligase” and “fibrin stabilizing factor” it circulates in plasma in a concentration of 20 microgram/ml. This protein exists in plasma as a tetramer comprised of two A subunits and two B subunits (A2B2). Each subunit has a molecular weight of approximately 80 000 Da, and the complete protein has a molecular weight of approximately 330 000 Da. The A subunits contain the transglutaminase catalytic site, whereas the B subunits have no catalytic activity but acts as a structural protein protecting the A subunits from clearance.
Factor XIII is dependent on thrombin and Ca2+ for activation. Thrombin cleaves on the C-terminal side of Arg37 in the A subunits, thereby releasing the activation peptide (residues 1-37 of the A subunit) and the B subunits and leaving the active form of the A2 dimer. The activated FXIII A2 subunit (FXIIIa) is the catalytically active form of FXIII.
The FXIII enzyme is the last enzyme to be activated in the coagulation cascade and functions to cross-link α- and γ-fibrin chains, resulting in a stronger clot with an increased resistance to fibrinolysis. Furthermore a number of antifibrinolytic, pro-haemostatic and adhesive proteins are cross-linked to the clot thereby providing a strong fibrin structure with increased mechanical resistance to the dissolution by plasmin and other proteolytic enzymes.
Commercially available purified or partially purified transglutaminase or Factor XIII preparations, in many cases, contain added stabilizers, such as human serum albumin (HSA). The use of protein stabilizers is problematic, since it decreases protein specific activity, increases the protein load when administered to patients, and may interfere with assessment of purity. It may also make the protein preparation immunogenic. These disadvantages of protein stabilizers make them particularly disadvantageous for stabilizing highly purified proteins, such as recombinant proteins. Additionally, use of protein stabilizers causes potential contamination with viral antigens when albumin, for example, is added.
FXIII, isolated from placenta or plasma or in the form of fresh frozen plasma, has been employed for many years for treatment of Factor XIII deficiency. These formulations, however, suffer from disadvantages of containing foreign proteins, with all the problems attached thereto such as unwanted immunogenicity.
It is now possible to prepare transglutaminases and Factor XIII using recombinant DNA technology. As used herein, rFXIII refers to recombinant Factor XIII. However, up to date there is no commercially available rFXIII product on the market.
The composition and activity of protein preparations used in therapy must be stable over relatively long periods of time. It is only rarely possible to achieve this stability in solution and, therefore, such products are frequently marketed in the dry state. Mild freeze-drying (lyophilization) is one of the methods of choice for drying such products. However, even when this method is used, stable preparations fulfilling the requirements for integrity and durability are difficult to achieve.
Freeze-drying of transglutaminase or Factor XIII solutions may for instance, for example, lead to a marked drop in activity and lose of purity. A further problem for freeze-drying of, for example, Factor XIII is created by a degradation product of Factor XIII the so called non-proteolytically activated species) (FXIIIa0), also referred to as pre-activated Factor XIII. FXIIIa0 is believed to pose a severe toxicity risk and injection of this may cause potentially undesirable side effects. It is therefore desirable to stabilize transglutaminase or Factor XIII formulations against formation of non-proteolytically activated products.
Various dry FXIII compositions are known in the prior art. Below are disclosed examples of disclosures of lyophilized FXIII compositions. WO2002/36155 describes that a lyophilized FXIII composition may be formulated with 1 mM EDTA, 10 mM Glycine, 2% sucrose in water, or with 20 mM Histidine, 3% wt/vol sucrose, 2 mM Glycine, and 0.01% wt/vol polysorbate, pH 8.0. U.S. Pat. No. 6,204,036 (Aventis Behring) concerns a stable lyophilized transglutaminase formulation comprising at least one additive selected from the group of D- and L-amino acids and salts, derivatives, homologs and dimers thereof, sugars and sugar alcohols, surface active ingredients, and reducing agents with the proviso that the additive is not glycine nor arginine, wherein the formulation does not contain a protein stabilizer and wherein said formulation is soluble without any turbidity in an aqueous solvent suitable for parenteral administration.
These formulations mentioned above, however may suffer from the disadvantage, described above, of high levels of non-proteolytically activated Factor XIII, which is considered an obstacle for clinical and commercial feasibility of these formulations. It is apparent, therefore, that stable dry transglutaminase and Factor XIII formulations with low levels of non-proteolytically activated products are greatly desired. Furthermore, such formulations should not require the addition of protein stabilizers, for example HSA.